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This study investigates the biocontrol potential of Trichoderma asperellum mutants against Rhizoctonia solani, Altaria altata, and Fusarium oxysporum and growth promotion of Populus davidiana × P. alba var. pyramidalis (PdPap poplar) seedlings. A T-DNA insertion mutant library of T. asperellum was constructed using Agrobacterium tumefaciens-mediated transformation. Sixty-five positive transformants (T1–T65) were obtained. Growth rates of the mutants T39 and T45 were the same, 39.68％ faster than the WT. In toxin tolerance tests, only T39 had greater tolerance to A. altata fermentation broth than the WT, but mutant T45 had the same tolerance as the WT to all fermentation broths. Furthermore, T39 and T45 had a greater antagonistic ability than the WT strain against R. solani and A. altata. The inhibition rate of the mutants T39 and T45 against A. altata are 73.92％and 80.76％, respectively, and 63.51％ and 63.74％, respec-tively. Furthermore, the three strains increased the activi-ties of superoxide dismutases, peroxidase, catalase (CAT) and phenylalanine ammonia lyase (PAL) in PdPap seedling leaves. CAT and PAL activity in the PdPap seedling leaves was 11.25 and 5.50 times higher, respectively, in the presence of T39 than in the control group and 12 and 6.35 times higher, respectively, in the presence of T45 than in the control group. All three strains promoted seedling growth and the root and stem development, especially mutant T45. Mutants T39 and T45 reduced the incidence of pathogenic fungi in poplar and stimulated poplar seedling growth.